Human granulocyte-macrophage colony-stimulating factor enhancer function is associated with cooperative interactions between AP-1 and NFATp/c

Area of Science:

• Molecular Biology
• Gene Regulation

Background:

• The human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter is regulated by an inducible upstream enhancer.
• This enhancer contains multiple binding sites for transcription factors, including Nuclear Factor of Activated T-cells (NFAT) and Activator Protein-1 (AP-1).

Purpose of the Study:

• To identify and characterize novel NFAT binding sites within the GM-CSF enhancer.
• To investigate the functional significance of these NFAT sites and their cooperative binding with AP-1 in regulating GM-CSF gene expression.

Main Methods:

• Deletion analysis to define the essential core of the enhancer.
• Methylation interference assays to determine DNA binding sites and consensus sequences.
• Electrophoretic mobility shift assays (EMSAs) to assess transcription factor binding and cooperativity.

Main Results:

• A novel NFAT binding site, GM420, was identified within the essential core of the GM-CSF enhancer.
• Three NFAT sites (GM330, GM420, GM550) demonstrated efficient function and cooperative binding with AP-1, resembling the interleukin-2 NFAT site consensus sequence GGA(N)9TCA.
• The GM170 site, with different spacing, supported independent binding but poor enhancer function.

Conclusions:

• Cooperative binding of NFAT and AP-1 to specific DNA motifs, with precise spacing, is crucial for efficient enhancer activity.
• The GM420 element plays a key role in the inducible regulation of the GM-CSF enhancer.
• Tight regulation of GM-CSF expression is achieved through the cooperative interactions of NFAT and AP-1 via independent induction pathways.